Amyotrophic lateral sclerosis (ALS) is a motor neuron disorder characterized by the degeneration and death of motor neurons. Currently there is no cure for ALS. Most ALS cases are sporadic. Only 5 to 10 percent of ALS cases are inherited. About 20 percent of familial ALS were caused by genetic mutations in superoxide dismutase 1 (SOD1). Mice expressing SOD1 mutants have been used frequently as models of familial ALS. In SOD1 mutants of different biochemical characteristics, we have shown endothelial degeneration in the spinal cord, microhemorrhages and blood-spinal cord barrier (BSCB) disruption before motor neuron degeneration. Treatment of SOD1G93A mice with a mutant form of activated protein C (APC) with minimal anticoagulant activity, designated as 5A-APC, slows disease progression and extends the lifespan and the symptomatic phase. We have shown that 5A-APC crosses the BSCB and transcriptionally downregulates SOD1 in motor neurons and microglia in SOD1G93A mice. 5A-APC also downregulates SOD1 in cultured N2a differentiated neurons expressing SOD1G85R or SOD1G37R mutants via protease activated receptors 1 and 3, which inhibits nuclear transport of the SP1 transcription factor. In SOD1G93A mice, 5A-APC eliminated microhemorrhages in the spinal cord and BSCB leakage and reduced microglial activation. Protein C (PC) is made as a single polypeptide precursor which undergoes a series of post-translational modifications (PTMs), including vitamin K dependent 3-carboxylation, to form a mature two-chain zymogen which circulates in plasma and can be converted to APC upon thrombin activation. The extensive PTM makes it very challenging to express high levels of recombinant PC in mammalian cells. In this application, two approaches will be employed to enhance the production of fully functional recombinant human 5A-APC in stable CHO cell lines. One approach is to coexpress vitamin K 2,3-epoxide reductase subunit 1 (VKORC1) to increase the efficiency of vitamin K recycling hence the enhancement of 3-carboxylation of 5A-PC. The other approach is to knock down endoplasmic reticulum (ER) stress/chaperone protein GRP78 to increase the efficiency of 5A-PC secretion. The overall goal of the Phase I study is to enhance production of fully functional 5A-APC mutant in mammalian cell system. Once proven successful, the high-producing cell line will be used to develop a manufacturing process in the Phase II study. Upon completion, the Company will seek internal funding to support 5A-APC GMP manufacturing, toxicity studies, IND-filing, clinical phase 1 and 2 studies in ALS patients. Phase 3 clinical study, NDA filing, and commercialization will be done through licensing and strategic partnering. PUBLIC HEALTH RELEVANCE: Currently, there are no effective therapies to prevent or reverse the course of amyotrophic lateral sclerosis (ALS). Recent findings indicated that the compromised blood vessels in the ALS-affected spinal cord and brain areas may play an important role in the disease progression. This application seeks to develop a potential new therapeutic designed to protect blood vessels as well as neurons in the ALS-affected regions.